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Abstract:

A system for imaging a fluorescently labeled sample is presented, The
system comprises a capsule, which is a closable structure made of a
material isolating the inside of the capsule from its surrounding
environment, and which has a support stage for receiving the sample and
carrying it thereinside during the imaging; and an optical device at
least partly accommodated inside the capsule and operable to illuminate
the sample with incident radiation to excite a fluorescent response of
the sample, detect the fluorescent response, and generate data indicative
thereof.

Claims:

1. A system for imaging a fluorescently labeled sample, the system
comprising a capsule which is a closable structure made of a material
isolating the inside of the capsule from its surrounding environment, and
which has a support stage for receiving the sample and carrying it
thereinside during the imaging; and a sensor arrangement accommodated
inside the capsule and operable to detect for sensing one or more
predetermined environmental condition inside the capsule, and comprises
inlet and outlet means for adjusting said at least one environmental
condition inside the capsule.

2. The system according to claim 1, wherein the light environment inside
the capsule during the imaging is sensed and maintained to be about 0.001
lux.

3. The system according to claim 1, wherein said sensor arrangement
comprises at least one temperature sensor.

4. The system according to claim 3, wherein said sensor arrangement
comprises at least one of the following: a light intensity sensor, an
electromagnetic radiation sensor, a gas sensor, and a humidity sensor.

5. The system according to claim 4, wherein said gas sensor is capable of
detecting the content of oxygen in the vicinity of the sensor.

6. The system according to claim 1, wherein said optical device comprises
a light source system, an image formation system, and light
directing/collecting optics having an objective lens arrangement and
defining illumination and detection channels of light propagation.

7. The system according to claim 6, wherein the light source system has a
plurality of light sources, each operable to generate the incident
radiation including a spectrum exciting a fluorescent response of the
sample.

8. The system according to claim 6, wherein said light
directing/collecting optics comprises a filter arrangement.

9. The system according to claim 8, wherein said filter arrangement
comprises a spectral filter assembly accommodated in the optical path of
light returned from the illuminated sample to select from the returned
light the fluorescent response and direct it to the detection channel to
propagate towards the image formation system.

10. The system according to claim 9, wherein said filter arrangement
comprises a spectral filter assembly in the optical path of the incident
radiation generated by the light source system to select the desired
exciting light and allow its incidence onto the sample.

11. The system according to claim 6, wherein said light
directing/collecting optics comprises a beam shaping assembly
accommodated in the optical path of the incident radiation generated by
the light source system.

12. The system according to claim 6, wherein the image formation system
comprises a detector arrangement.

13. The system according to claim 12, wherein the detector arrangement
comprises a single detector.

14. The system according to claim 13, wherein said single detector is a
CCD camera or a CMOS camera.

15. The system according to claim 14, wherein said single detector is a
cooled system.

16. The system according to claim 12, wherein said detector arrangement
has at least two different detectors.

17. A system for imaging a fluorescently labeled sample, the system
comprising:a capsule, which is a closable structure made of a material
isolating the inside of the capsule from surrounding environment, the
capsule comprising a support stage for receiving the sample and carrying
it thereinside during the imaging process in a manner enabling
displacement of the sample with respect to an inspection plane, and
having inlet and output channels operable to affect environment
conditions inside the capsule;an optical device at least partly
accommodated inside the capsule and operable to illuminate the sample
with incident exciting radiation to excite a fluorescent response of the
sample, detect the fluorescent response, and generate data indicative
thereof;a sensor arrangement accommodated inside the capsule and operable
to detect at least two of the following environment conditions inside the
capsule: temperature, light intensity, electromagnetic radiation
intensity, content of at least one gas in the capsule, and humidity; and
to generate data indicative thereof; anda control unit connectable to the
capsule and response to said data indicative of the detected fluorescent
response to output an image of the illuminated region of the sample,
responsive to said data indicative of the at least one environment
condition to operate the inlet and output channels of the capsule so as
to provide a desired environment condition inside the capsule.

Description:

FIELD OF THE INVENTION

[0001]This invention is generally in the field of optical
measurement/inspection techniques and relates to an optical system and
method for inspecting fluorescently labeled biological specimens.

BACKGROUND OF THE INVENTION

[0002]One of the new emerging techniques used today in the research of
molecular biology and genetics is fluorescent labeling of a biological
specimen. According to this technique, fluorescent probes are used to
mark the specific locations in a biological specimen aimed at detecting
different genes, chromosomes, DNA strands, proteins, and bacteria.

[0003]In recent years, the fluorescent labeling based techniques have
started to push their way into the diagnostic world, and it is
anticipated that in the near future diagnostic assays based on
fluorescent labeling will be used more and more routinely.

[0004]According to conventional techniques, the detection of fluorescent
probes is done in research laboratories by using an "off the shelf"
fluorescent microscope. The use of a fluorescent microscope was a logical
choice, since this machine was readily available in most research labs.
Furthermore, it was a familiar tool to all researchers, and had the
benefit of being a multi purpose platform used for other lab applications
as well.

[0005]However, the detection of fluorescent probes in a biological sample
by means of the conventional fluorescent microscope suffers from several
drawbacks associated with the following. Today, in diagnostic
laboratories that use fluorescent techniques, an operator with genetic
training typically manually operates a fluorescent microscope. The
operator must manually select the correct objective and filters, manually
scan the slide and search for good genetic material, focus on each image,
analyze the fluorescent signals, and write down his analysis. The
operator has to look through a binocular eyepiece during the entire
process, which is a cumbersome and tiring process. Thus, an operator
cannot work on the microscope for more than a few hours continuously, and
not more than 8-10 hours daily. This of course limits the number of tests
a lab can perform, thus limiting the lab's throughput significantly.

[0006]Furthermore, the laboratory, where this analysis is done, has to be
in blackout conditions. This is associated with one of the major problems
of using fluorescent labeling for routine diagnostic assays, consisting
of keeping the fluorescent labeling "alive" long enough to finish the
entire procedure, which typically includes scanning the sample on a
slide, looking for region of interests (ROIs) in the sample (for example,
a nucleus of the cell or a chromosome), focusing on the ROIs, taking an
image thereof, refocusing on sub areas within the ROI (for example
labeled genes), and taking images of the these sub areas as well. This
procedure takes quite a while, since a large number of ROIs must be
considered to achieve the high reliability required from an assay used
for diagnostic purposes. For example, in prenatal FISH tests
(fluorescence in situ hybridization) at least 100 good regions of
interest (nuclei) are needed to be imaged for giving a reliable diagnosis
from the test ("Prenatal diagnosis using interphase fluorescence in situ
hybridization (FISH)", Prenat Diagn 2001; 21: 293-301. DOI: 10.1002/p.
57). FISH method is typically used to detect the absence or excess of a
specific gene (e.g., elastin gene) from a chromosome, e.g., to detect the
presence of down syndrome.

[0007]To detect 100 good enough regions of interest, one must scan several
hundreds of fields on the sample. Working for so long on the sample
raises the problem of bleaching. Bleaching of a sample causes the
fluorescent probes to fade, thus making the reading of the sample
impossible. This phenomenon, which occurs within minutes, is stimulated
by light and oxygen. Operation with the conventional fluorescent
microscope thus requires operation in the dark, and implies that other
activities requiring light cannot be carried out at the same time and
place, when fluorescent analysis is in process. As a result, all
laboratory work has to be halted when fluorescent signals are analyzed,
or a separate room has to be assigned for the fluorescent microscope.
Furthermore, the necessity to work in a dark environment, affects the
performance of the microscope operator. Working in the dark, is no doubt,
a cumbersome task.

[0008]Other environmental hazards of the conventional techniques, such as
heat, humidity, radiation, electromagnetic waves, also have undesired
influence on some biological samples. With the conventional microscope
and conventional technique, operating personnel are exposed to safety
hazards due to UV light typically used to excite the fluorescent sample,
but is harmful to people.

[0009]The use of a "semi-automatic" fluorescent microscope set-up has been
proposed (BX51 Epi-Fluorescence Microscope commercially available from
Olympus). In this set-up, a digital camera and a computer are added to
the fluorescent microscope solely for archiving the images so as to
enable reviewing the images at a later time. An "automatic" fluorescent
microscope set-up (DM RXA2 Fluorescence Microscope commercially available
from Leica) allows for integrating the "off-the-shelf" components such as
a microscope, digital camera, scanning stage, and computer, in
conjunction with a software package that controls the operation of these
components. However, using the "off-the-shelf" components that were not
designed specifically for fluorescent diagnostic tasks (to comply with
the demands of the fluorescent-based diagnostic world) obviously
decreases performance and increases costs of the optical system.

SUMMARY OF THE INVENTION

[0010]The use of fluorescent probes routinely for diagnostic purposes
creates new standards and demands for a fluorescent detection system.
Such a system must provide a complete and full solution for a
fluorescent-based diagnosis. The system must be characterized by a high
throughput capability, high levels of automation, a simple graphic user
interface (GUI) that enables minimal user operating mistakes and thereby
allows for layman operation of the system, a high level of reliability
and accuracy, and last but not least, it must be economically beneficial.

[0011]The main idea of the present invention consists of solving the above
problems by the encapsulation of a stage intended for supporting a
fluorescently labeled sample under inspection, and an optical inspection
(imaging) system. Such a capsule is made of a material preventing the
penetration of light into the capsule from the outside thereof (i.e.,
non-transparent material), and preferably also preventing the penetration
of electromagnetic waves (i.e., electrically conductive material). The
capsule preferably also includes one or more environment control sensors,
and is equipped with means enabling the adjustment of the corresponding
environment conditions inside the capsule so as to meet the requirements
of an optical inspection of fluorescently labeled samples.

[0012]The technique of the present invention thus provides a unique
platform that complies with the new demands arising from turning the
latest research techniques in fluorescence into tomorrow's diagnostic
tools. The system and method of the present invention makes up a
Fluorescent Working Station (F-WOS) constructed and operated to provide
optimal conditions for acquiring high-end fluorescent images (i.e., the
combination of low light sensing, small size and high magnification,
together with the enhanced image processing and control) from biological
samples for research and diagnostic tasks, and to provide optimal
conditions for the sample, fluorescent signals, operator, and all other
personal working in the lab. By designing a complete system especially
for fluorescent diagnostics, the high performance, high throughput, and
low price system is obtained. It should be noted that fluorescent
signals, for diagnostic tasks, are characterized by a low light (about
0.001 lux), capability of detecting small size fluorescent labels
(0.1-0.4 microns), fast fluorescent intensity decay with time
(bleaching), and environmental sensitivity. This means that in
fluorescent imaging, working on the "edge" of technology is required, and
it is thus crucial to address the fluorescent signal quality issues, as
well as the imaging quality issues, for obtaining the best results.

[0013]The F-WOS of the present invention is the first fluorescent-based
system that makes a significant effort in improving the quality of the
fluorescent signals, and not just improving the quality of the acquired
images. The F-WOS enables to easily load a fluorescent slide into the
capsule, automatic scanning of the slide in the X,Y,Z planes at the
appropriate resolution and speed, searching for the designated regions of
interest on the fly, and optimal acquisition of the required images for
research and diagnosis needs.

[0014]According to one aspect of the present invention, there is provided
a system for imaging a fluorescently labeled sample, the system
comprising a capsule, which is a closable structure made of a material
isolating the inside of the capsule from its surrounding environment, and
which has a support stage for receiving the sample and carrying it
thereinside during the imaging; and an optical device at least partly
accommodated inside the capsule and operable to illuminate the sample
with incident radiation to excite a fluorescent response of the sample,
detect the fluorescent response, and generate data indicative thereof.

[0015]Preferably, the capsule comprises one or more sensors for sensing
the environment condition(s) inside the capsule to be controlled, and
inlet and outlet means enabling to desirably affect the corresponding
condition(s) inside the capsule. The capsule thus presents a "controlled
environmental capsule" (CEC). The sensors suitable to be used in the
capsule include at least one of the following: a temperature sensor, an
ambient light sensor, an electromagnetic radiation sensor, oxygen or
other gases' sensor, and a humidity sensor. The CEC is designed to
protect the biological media and the fluorescent probes therein from
environmental hazards, and to provide them with optimal conditions during
the imaging. For example, the capsule protects the sample from unwanted
light in the room, high temperature, and the presence of oxygen, causing
the sample to fade quickly (the Bleaching phenomena). Providing these
optimal conditions for the fluorescent sample improves the quality of the
fluorescent signals. The CEC also provides protection for the operating
personal from hazardous conditions of the system such as the use of UV
light. Furthermore, the encapsulation of the sample with the optical
device enables installation of the working station in any room or
laboratory, without the necessity to darken the room when working with
the fluorescent signals, thus stopping all other activities in the
laboratory at that time.

[0016]The optical device provides: means for selecting and guiding the
excitation light to the sample, means for collecting and selecting the
desired emitted (excited) light from the sample, means for forming the
fluorescent image at a selected focal plane, and preferably also means
for enlarging the images. All components are designed to obtain the best
fluorescent images possible.

[0017]The optical device thus includes a light source system, an image
formation system, and light directing/collecting optics. The light source
uses one or more light sources of the kind generating excitation incident
radiation to excite a fluorescent response of the sample. The optics used
may include a light guiding means (filters) for selecting and guiding the
desired excitation light to the sample, a beam shaping optics in the
optical path of the exciting light, a light collecting optics for
collecting light coming from the sample and selecting therefrom the
desired fluorescent light, and an imaging optics to form the fluorescent
image of the sample. The detection unit may include one or more detectors
(e.g., with different specifications). For example, the detection unit
may include a single imaging detector, but preferably includes at least
two such detectors with different attributes. For example, one detector
is a self-designed CMOS camera aimed at identifying pre-defined ROI(s)
on-the-fly (in real time) and processing the images on the camera chip
itself, thereby saving the need to send the images to an external
computer, and the other detector is a cooled CCD camera aimed at
providing high quality images for analysis and acquisition.

[0018]The device further includes a scanning system, which enables
scanning the sample at different resolution and speeds at the X, Y and Z
directions (3D scanning). The scanning system supports the ROI search and
identification, preferably utilizing also auto-focusing abilities, and
provides the F-WOS with the ability to automatically detect a large
amount of fluorescent signals in a short period of time.

[0019]The system of the present invention utilizes a control unit
(Computerized Central Control) that automatically controls and
synchronizes the operation of the entire workstation). The control unit
receives data from data indicative of the detected fluorescent response
from the detector(s) to generate data indicative of an image of the
sample, and preferably also receives data indicative of the environment
condition(s) inside the capsule to analyze this data and operate the
inlet and output channels of the system accordingly. The control unit is
typically a computer device connectable to the capsule through wires or
wireless communication, and includes inter alia a database utility for
storing a specifically designed database, a data processing and analyzing
utility preprogrammed with specially designed algorithms, and a display
unit. The control unit preferably comprises appropriate communication
means enabling "downloading" of the acquired images and all relevant data
to the database. The algorithm packages especially designed for use in
the F-WOS are responsible inter alia for the following: finding
predefined regions of interest (ROI), focusing on these regions,
analyzing the images in the ROI, selecting sub areas in the ROI for
further acquiring and analysis, calculating the optimal parameters (i.e.
roundness, overlapping, size and some others) for acquiring the different
images, and giving a diagnosis evaluation based on the acquired images
and predefined statistics. The same or an additional control unit is
operable to automatically control and synchronize the operation of the
entire workstation. The database utility enables the following
operations: saving the acquired images, saving all other relevant
information, conducting different search algorithms on the database, and
options for adding new fields of data to each item in the database.

[0020]The fluorescent sample may be of the kind prepared in the FISH
method, and may be used for obtaining diagnostic results, e.g., by using
Aneuploidy methods.

[0021]According to another aspect of the present invention, there is
provided an optical system for imaging a fluorescently labeled sample,
the system comprising: [0022]a capsule, which is a closable structure
made of a material isolating the inside of the capsule from surrounding
environment, the capsule comprising a support stage for receiving the
sample and carrying it thereinside during the imaging process in a manner
enabling displacement of the sample with respect to an inspection plane,
and having inlet and output channels operable to affect environment
conditions inside the capsule; [0023]an optical device at least partly
accommodated inside the capsule and operable to illuminate the sample
with incident exciting radiation to excite a fluorescent response of the
sample, detect the fluorescent response, and generate data indicative
thereof; [0024]a sensor panel accommodated inside the capsule and
operable to detect at least one of the following environmental conditions
inside the capsule: temperature, light intensity, electromagnetic
radiation intensity, content of oxygen or other gases, and humidity; and
to generate data indicative thereof; and [0025]a control unit connectable
to the capsule and response to said data indicative of the detected
fluorescent response to output an image of the illuminated region of the
sample, responsive to said data indicative of the at least one
environmental condition to operate the inlet and output channels of the
capsule so as to provide a desired environment condition inside the
capsule.

[0026]According to yet another embodiment of the invention, there is
provided a method of imaging a fluorescently labeled sample utilizing the
above system, the method comprising: [0027](i) first scanning of the
sample to detect regions of interest in the sample and determine
coordinates of the detected regions of interest; [0028](ii) utilizing the
results of the first scanning, and performing second scanning of the
regions of interest to detect sub-areas of the regions of interest
containing the fluorescent labels and acquire images of said sub-areas.

[0029]The first scanning is performed at low oxygen level in the capsule
as compared to that of environment outside the capsule, and relatively
low temperature of the sample as compared to that of the second scanning.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]In order to understand the invention and to see how it may be
carried out in practice, a preferred embodiment will now be described, by
way of non-limiting example only, with reference to the accompanying
drawings, in which:

[0031]FIG. 1 is a schematic illustration of a fluorescent optical system
according to the invention;

[0032]FIG. 2 more specifically illustrates as optical device suitable to
be used in the system of FIG. 1; and

[0033]FIG. 3 exemplifies a flow diagram of the operational steps of a
method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0034]Referring to FIG. 1, there is schematically illustrated an optical
system 10 according to the invention for imaging fluorescently labeled
samples. The system 10 comprises a control environment capsule (CEC) 12
that is formed with a door 14 for receiving a sample-on-slide S and
includes a stage 16 for supporting the sample S during the system
operation, and an optical device 18. The sample is a biological specimen
that should be prevented from being illuminated all the time except for
that needed for imaging the sample. To this end, the sample-on-slide may
be stored in a "pre-loading" cassette designed for carrying a single
slide or multiple slides in darkness. The slide can be manually loaded
onto the stage 16, or automatically by means of a robot. This can be
implemented by an automatic movement of the stage 16 into a loading
position when the door 14 is opened. If the slide in cassette is used,
the cassette can be formed with a shiftable cover that is automatically
shifted into its open position, when the system is put in operation.

[0035]The optical device 18 comprises an excitation light system, an image
formation (detection) system, and light directing/collecting optics, as
will be described more specifically further below with reference to FIG.
2. In the present example, all these elements of the optical device are
located inside the capsule 12, but it should be understood that either
the light source or detector(s) or both may be located outside the
capsule and light may be guided towards and/or away from these elements
using optical path or fibers.

[0036]The CEC 12 preferably also has a sensor arrangement (e.g., sensor
panel) 20 including one or more sensors capable of measuring the current
environment condition(s) in the capsule. The sensor arrangement 20 may
include, for example, light sensors, oxygen sensor, temperature sensor,
humidity sensor, and a pressure sensor.

[0037]Connectable to the system 10 (through wires or wireless) is a
control unit 22 that operates the system and processes data indicative of
images of the sample.

[0038]The CEC 12 is a closed chamber made of non-transparent electrically
conductive material (e.g., metal), which isolates the sample from all
undesired conditions such as light, air, electro-magnetic radiation,
temperature, and humidity, and has inlet and outlet channels 24 and 26
through which the environment condition(s) inside the CEC can be
appropriately adjusted. The inlet channels 24 are responsible for
entering matter to the CEC to balance the environment. For example,
cooled air can be entered through the inlet channels to thereby lower the
temperature in the capsule, when a temperature level higher than a
desired one is detected via a respective sensor. The outlet channels 26
are responsible for drawing out matter from the capsule environment. For
example, drawing out air through the channel 26 and entering nitrogen
through the inlet cannel 24 would cause lowering of the oxygen content in
the capsule.

[0039]The sample-on-slide S is loaded into the chamber through the door
14, and put into its inspection position on the support stage 16. The
latter is driven by a suitable drive (not shown) for movement along the
X, Y, and Z axes. It should be understood that in the present example of
the invention, where auto-focusing is implemented as a passive
mathematical procedure utilizing grabbing of several pictures along the
Z-axis, and then calculated the focal position, the stage 16 is the only
movable element. Generally, however, any other suitable auto-focusing
technique can be used utilizing the Z-movement of an objective lens.
Prior to loading the sample into the CEC, the desired environment inside
the CEC is set. This can be done using a gauges interface 28 or from the
control unit 22. It should be noted that the gauges interface can be part
of the CEC, of the control unit 22, or can be a separate unit connectable
to the CEC and to the control unit 22. The control unit 22 has a database
utility 23, a data processing and analyzing utility 25, and a display
utility 27. It should be noted that, generally, the database utility can
be part of another external device connectable to the control unit.
Preset environmental conditions, which are optimal for a specific sample
under inspection, may be automatically loaded from the database utility.
In other cases, manual setting of the environmental parameters may be
desired. The environmental data generated by the sensor(s) 20 is
transferred to the control unit 22 via a communication channel
(wire-based or wireless), and is processed by comparing this data to the
set parameters. The desired environment is then obtained by controlling
the inlet and outlet channels 24 and 26 in a closed loop with feedback
readings from the sensors 20 or in an open loop (by manual operation of
the inlet and outlet channels. For example, if due to some light heat the
temperature in the capsule is changed, the respective sensor feels this
change, and generates a signal to the control unit thereby initiating a
cooling procedure, until the temperature reaches the desired level.

[0040]FIG. 2 more specifically illustrates the optical device 18. The
device 18 comprises an excitation light source system 30; an image
formation system 32 including a detector unit; a light
directing/collecting optics 34; and a scanning system 35. As shown in the
figure in dashed lines, the light source 36' and a detector unit 32' (or
one of them) can be located outside the CEC, in which case light is
guided through optical path or fibers F. Generally, the system of the
present invention can utilize the conventional fluorescent microscope
modified by placing at least its light directing/collecting optics into
the CEC equipped with a sensor arrangement and inlet and outlet channels.

[0041]The sample is a biological specimen having fluorescent labels.
Accordingly, the excitation light system 30 includes a light source 36 of
the kind generating light including a wavelength range λ1
capable of exciting a fluorescent response λ2 of the sample.
Such a light source may be of any suitable type for fluorescent imaging,
for example, a Mercury light source, a Xenon light source, a laser-based
light source generating light of UV, Visible, or IR, or a combination of
these light sources. The light source system may include: a single light
source emitting light of a desired spectrum capable of exciting a
fluorescent response of the sample, a single broadband light source
associated with one or more spectral filters for selectively separating a
desired exciting component from the emitted light, or more than one light
source of different spectral ranges or of the same spectral range, each
associated with an spectral filter.

[0042]The light directing/collecting optics 34 typically includes an
objective lens arrangement 39, which is preferably used for both focusing
the incident exciting beam onto the sample and collecting light returned
from the sample. As shown, in the present example, the light
directing/collecting optics utilizes a light filtering assembly 40
accommodated in the illumination channel, namely, in the optical path of
light generated by the light source system. The assembly 40 is designed
to select the desired wavelength for the excitation light. Several
different filters can be located on a wheel or a slider, so they can be
easily changed within and between applications. This configuration gives
the system the ability to easily handle many different fluorescent
applications. The filtering arrangement preferably comprises a further
filter assembly 42 in the detection channel, namely, in the optical path
of light propagating towards a detector to enable detection of the
desired wavelength. Similarly, the filters may be located on a wheel or a
slider, so they can be easily changed within and between applications, to
thereby allow for handling many different fluorescent applications. The
image formation system uses the objective lens arrangement 39 to form
from the selected fluorescent light an image of a desired size on a
predefined focal plain of the lenses. This stage is important, when
infinity optics is used along the optical path.

[0043]In the preferred embodiment of the invention, the excitation and
image formation systems operate in the epi-fluorescent fashion. In this
case, the microscope objective (generally, focusing optics) 39 is used
for guiding the exciting light λ1 to the sample as well as for
collecting the emitted fluorescent light λ2 from the sample,
and enlarging it. To this end, a dichroic mirror 46 is used to spatially
separate between the excitation and excited light and direct them along
the illumination and detection channels, respectively. The mirror 46 and
the filter assemblies 40 and 42 can be arranged as a common filter cube.
The use of epi-illumination conditions eliminates the need for a
transparent slide.

[0044]The image formation system 32 is designed for replacing the viewing
of the image by a human eye at the focal plain. A light detector
arrangement is used to automatically capture the images instead of the
human observer. An algorithm, which calculates the best parameters for
each image, is used for this procedure. Generally, a single detector (CCD
or CMOS camera) can be used for the purposes of the present invention,
but preferably the detection system uses multiple light detectors
(preferably two or three such detectors). In this case, each light
detector can be of a different type, thus providing different attributes
to the detected images. The detectors may differ in resolution,
sensitivity, noise, and the overall image quality they give. In one
specific embodiment of the invention exemplified in FIG. 2, the image
formation system 32 includes two light detectors D1 and D2.
Detector D1 is a high performance detector, preferably CCD camera
(generally, CMOS can be used as well), which is used for catching the
fine details of the image. A high-performance cooled CCD camera is
capable of delivering quality low light, high-resolution images. The
other detector D2 is preferably a CMOS camera (or CCD) is used to
provide fast images for evaluating the focus plane and detecting regions
of interest "on the fly" (i.e., regions having fluorescent singles or
labels) This second detector D2 (CMOS camera) also provides the
system with fast hardware components that can perform some image
processing on-the-fly, without the need to transfer the entire image to
the computer. The two detectors are thus used for, respectively, rough
estimation of the ROI, and detecting and analyzing the fluorescent
signals (labels) themselves in sub-areas of ROIs (which requires higher
magnification)

[0045]The scanning system 35 includes a drive mechanism for moving the
stage with a sample-on-slide in the X-Y plane (inspection plane) and
along the Z-axis. The scanning system is appropriately operated by the
control unit to enable a 3-D search, looking for relevant predefined
information on the fluorescent sample. The search is conducted by moving
the sample (stage) in the x, y, and z directions at different resolutions
and speeds, and searching for predefined objects. In the F-WOS system,
the predefined objects are cell nucleus, chromosomes, or specific labeled
genes. The scanning system follows a predefined search route, and saves
the search results, which include the presence of the searched object,
i.e., region of interest, as well as its exact coordinates. The system is
also capable of returning accurately to the certain ROI using the saved
coordinates. The searching speed, resolution, path, and objective
location are calculated, using predefined information specifying the
sample type, and special algorithms. The entire search is supported by
advanced image processing algorithms in both hardware and software. The
scanning system provides the F-WOS with the ability to automatically
detect and acquire a large amount of fluorescent signals in a short
period of time.

[0046]Turning back to FIG. 1, the control unit 22 operates to provide
automatic monitoring, controlling and synchronizing of the operations of
all functional elements of the system 10 (working station). The control
unit enables to load pre-set programs for scanning in a predefined manner
according to a specific application, as well as to easily program the
station for new scanning modes and applications. The communication
between the system 10 and the control unit 22 provides for the continuous
flow of data and information from the different stages of the system to
the control unit and back from it to the different stages of the system.
All the acquired images as well as the system parameters and other
selected parameters (such as exposure time, gain, sample's initial
location, coloring materials, etc.) are transferred to the control unit
22 via the communication channels that can be wired channels utilizing
any of the known protocols for image and data transfer (such as
"FireWire"), or can be wireless communication channels utilizing
broadcasting techniques to transfer the information to the control unit
and back (for example the known BlueTooth technology). As indicated
above, the control unit has the data processing and analyzing utility
preprogrammed with specially designed algorithms for the FWOS. These
algorithms are responsible inter alia for the following functions:
finding predefined regions of interest (ROI), analyzing the images in the
ROI, selecting sub-areas in the ROI for further acquiring and analysis,
calculating the optimal parameters (i.e. roundness, overlapping, size and
some others) for acquiring the different images, selecting the best
configuration for the light source, calculating the preferred optical
path for the sample, and providing a diagnostic evaluation based on the
acquired images and predefined statistics.

[0047]The database utility 23 is designed to enable the following
functions: saving the acquired images as well as all other relevant
information including among other things: all systems parameters, slide
(sample) information, patient information, and analysis results. The
control unit also offers advanced search algorithms to use on the
database utility on both images and data, and an easy option for adding
new fields of data to the database. The Database can be local
one--physically residing on the control unit, or it can be physically in
a far a way location. Access to a central database can be through the
Internet or any other type of net. Since the database includes sensitive
information, it must include some kind of security facilities (personal
key or password) providing authorization control in both writing and
reading from the database. Since the F-WOS system of the present
invention is intended for use in hospital labs, the database format is
compatible with other computerized databases in the hospital.

[0048]It is important to note that the system of the present invention
provides for an improved GUI of the system. Basically, the operation of
the system is carried out from the control unit 22 (e.g., personal
computer). The operator will first have to identify himself and enter the
ID of the sample. Then, he will be able to select the name of the
fluorescent dye used for labeling the sample, or the fluorescent
diagnostic kit used from a set of preset programs, thus defining the
sample type. Based on this information, the system will be able to set
all its parameters automatically and start operating. The selection of
all parameters includes the selection of the correct objective, filters,
scan type, resolution, image acquisition parameters, temperature, light
intensity, scanning route, etc. When a new fluorescent dye is used, the
user can manually set these parameters from the GUI, and from that point
the system operates automatically. After all images are acquired, the
operator can view the images as an image gallery on the monitor of the
control unit. Using the image gallery tools, the operator can easily and
quickly view all images, and complete the diagnostic procedure.

[0049]The system operation thus starts with setting the desired
environment inside the CEC, and loading a fluorescent sample-on-slide
into the CEC. Then, the sample type is set. To this end, the operator
enters all relevant information (slide ID, operator ID etc.) and sets the
sample's type, either by selecting it from a pre-set menu or by setting
it manually. The process ends, when a complete set of digital images is
stored in the database facility for further analysis. To achieve this,
the system utilizes a two-step scanning scheme. This scheme will now be
described with reference to FIG. 3.

[0050]A fluorescently labeled sample is entered into the system (i.e.,
into the capsule), and the sample's type is set. Then, a calibration
process based on the input parameters is executed. During this process, a
set of images is acquired under different conditions such as light
intensity, filter type, and Z-axis position. From this process, the
optimal parameters are chosen. For example, the optimal light source
intensity is determined as that corresponding to an optimum between the
maximum fluorescent emission of the sample obtained at the minimum
bleaching. Using the calibration results and all a priori information
entered to the system, all optical parameters, such as objective, light
source intensity, filters, optics, are set automatically for the first
scan. To enable optimizing of the results of the first scan, all
environmental parameters (e.g., temperature, oxygen level) are also set.
For the first scan, a low temperature (about 2-8 centigrade, i.e.,
substantially frozen or slightly heated sample) and low oxygen level (as
compared to that of the environment outside the CEC) is used. For the
entire system operation (i.e., the first and second scans), dark
environment inside the CEC (for example, less then 0.001 lux) is set and
kept, except for the time period of the light source operation). The
calibration results and all a priori information entered into the system
are also used to calculate the route of the scanning process, such as to
provide the desired images in minimum time, with minimum bleaching.

[0051]The first scan consists of a quick screening of the slide's content,
so as to identify the ROIs as quickly as possible, with minimum bleaching
to the sample. The ROIs are those regions in the sample that contain cell
nuclei or chromosomes during the Meta-Phase (i.e., containing fluorescent
labels and preferably of a desired size and roundness). Also determined
in the first scan is overlapping between the adjacent ROIs, so as to take
this information into account for farther data analysis. For this
screening process, a 3-D search is conducted on the slide (sample). Since
a screening process is carried out at this stage, rather than the image
acquisition process, and since the fluorescent images sought at this
stage are relatively large (an area of about 4 microns instead of 0.2
microns considered for the image acquisition process), a special scanning
mode is used, aimed at providing a maximal speed and minimal bleaching,
at the expense of image quality. This is achieved by working with a low
temperature, low resolution, fast and relatively low image-quality scan.
The term "low temperature scan" means that the slide is kept at a low
temperature. This may be achieved by cooling the entire capsule space, or
by using a local cooling device, like a paltrier, on the slide itself.
The use of a low temperature is known for slowing down a physical
process. This means that by keeping the slide cold, both the bleaching
and the fluorescent processes are slowed down. Operation with a correct
temperature provides the system with decreased bleaching and a detectable
image. As for low resolution and fast processing, this can be achieved by
using CMOS digital camera for the first scanning. This camera provides
sufficiently good images in a short time. Using the CMOS camera will
enable obtaining lower resolution images at a faster rate, compromising
the image quality and increasing speed. Furthermore, the CMOS camera used
in the present invention includes hardware capable of performing ROI
identification algorithms on-the-fly (in real time). The use of such a
set-up, in addition to speeding the identification algorithm by using
hardware instead of software, eliminates the need to transfer hundreds of
Mega pixel size images to the control unit, in order just to detect
whether the scanned field includes any ROI or not. After the hardware
identification algorithm is executed on the image, the only data that
needs to be transferred to the control unit for further use in the next
scan is data indicative of the X, Y, Z coordinates of the detected ROIs.

[0052]Now, the second scan is performed. Similarly, using the calibration
results and all a priori information entered into the system, all optical
parameters and acquiring parameters, are set automatically for the second
scan, and in order to optimize the results of the second scan, the
calibration results and all a priori information entered to the system
are used to set all the environmental parameters (temperature, Oxygen
level). For the second scan, a relatively high temperature
(10-20°), the same low oxygen level, and dark environment are set.
Using the results of the first scan (i.e., the coordinates of all
detected ROIs), the route of the scanning process is calculated. This
route has to provide the system with the best sequence, which will track
all the ROIs detected in the first scan, with minimum bleaching in
minimum time. The second scan is aimed at going to the ROIs detected in
the first scan, searching for fluorescent signals (labels) containing
sub-areas in these regions, and acquiring images of these sub-areas (by
illuminating them with suitable exciting light and collecting fluorescent
response). To this end, a fine 3-D search in these ROIs is used. Since
the fluorescent labels can be very faint and small (about 0.2 microns),
optimal conditions should be used for this scanning process, as compared
to those of the first scan. Furthermore, this second scanning process
includes image acquisition for diagnostic purposes, thus, top-quality
images are required. To satisfy these requirements, a top-quality cooled
CCD camera is used. The camera is operated at its top performance: no
binning and a slow readout. The environmental parameters are also set to
give maximum fluorescent signals, even at the expense of bleaching. To
achieve this, the slide is heated to provide maximum fluorescence. This
process is continued until the number of detected ROIs with signals (with
fluorescent labels) reaches a predefined threshold. Thus, the information
obtained with the second scan includes images of the detected fluorescent
labels containing sub-areas found in the ROIs detected in the first scan,
and the X,Y,Z coordinates of the ROIs, where signals were found.

[0053]During the above process, the images of the fluorescent signals
(labels) and the cell nucleus (or chromosomes) are acquired separately.
After all images are acquired, the matching nucleus and signal images are
combined to obtain the final color image. Special image enhancement
techniques may be used at this stage, to further improve the images. This
final set of images is the image set used for further diagnostic purposes
by the genetic expert.

[0054]The present invention thus provides the optical system and method,
consisting of encapsulation of the sample-on-slide and the optical
device, build up the fluorescent working station (F-WOS), presenting a
complete solution for automatic scanning of biological fluorescent
samples. The CEC provides optimal working conditions by encapsulating the
sample and the optical device, and preferably also environment sensor(s),
which provides for automatically monitoring and controlling environment
conditions. One of the advantages of the encapsulation of the F-WOS is
that the sample is protected from ambient light in the room, thus
bleaching is significantly reduced. The CEC can also provide the sample
with an environment free of oxygen, again reducing the bleaching effect
significantly. Thus, the CEC provides an environment which enables longer
scans of the sample without bleaching. Other environmental hazards such
as heat, humidity, radiation, electromagnetic waves, that may also have
undesired influence on some biological samples, can also be avoided, when
the sample is protected in the CEC. The encapsulation also provides
protection of the electronic devices (for example camera), by shielding
the electronic devices from electromagnetic noise in the room. An
additional benefit of using the CEC is the significant improvement in
signal to noise ratio of the acquired images. This is done by keeping the
entire optical system isolated from its surroundings. Thus, ambient light
in the room and other electromagnetic noise are shielded from the system.
Another important advantage of the CEC, is its ability to protect the
operating personal from hazardous condition of the system such as UV
light. Additionally, by encapsulating the system, it can be used in any
laboratory, without the necessity of providing blackout conditions for
the entire laboratory, thus also enabling other people and system
operation in the same space with the F-WOS without disturbing each other.

[0055]Those skilled in the art will readily appreciate that various
modifications and changes can be applied to the embodiment of the
invention as hereinbefore exemplified without departing from its scope
defined in and by the appended claims.